Analysis of cancer cells has led to the discovery of more than 500 tumor-specific chromosome aberrations. Detailed analysis of the breakpoints involved in these structural chromosomal rearrangements has been instrumental in the discovery of many cancer-related genes. Of all possible types of structural chromosome anomalies, inversions, which represent a reversal of orientation of a DNA segment within a chromosome, are found comparatively rarely among the known tumor-specific aberrations. Inversions can have genetic effects similar to the easily detected translocations between different chromosomes seen in cancer. Both can result in effects such as disrupting regulatory sequences that control gene expression or creating genetic rearrangements like gene fusions. Inversions form through the same mechanism as translocations, the misrepair of DNA double-strand breaks. Thus, it might be expected that translocations and inversions should be found in comparable numbers. One possible explanation for the discrepancy is that standard karyotype analyses are relatively insensitive to the detection of inversions and consequently have largely failed to find many tumor-specific chromosome aberrations of this type.
New approaches to measuring incorrect rejoining of radiation-induced DNA double-strand breaks in human cells has led to the conclusion that radiation produces at least ten times the number of chromosomal rearrangements than can now be observed cytogenetically, the vast majority of which are intra-chromosomal (that is, small interstitial deletions and inversions). To the extent that radiation is representative of other mechanisms of creating inversions, it appears likely that their significance has been underestimated and underappreciated in many diseases in addition to cancer.
In addition to cancer cytogenetics (the study of chromosomes and how changes in chromosome structure and number can lead to the loss of regulation and control of cell proliferation, and orderly differentiation of cells in tissues), chromosome analysis is widely used in prenatal screening as well as the diagnosis of congenital abnormalities, learning difficulties, impaired fertility, and sexual development problems.
The two methods frequently used for detection of gross cytogenetic aberrations such as translocations are whole chromosome painting by fluorescence in situ hybridization (FISH), and G- or R-banding. The sequence does not have to be known for either technique. Both chromatids of a chromosome are indiscriminately targeted by these techniques. Whole-chromosome-specific-FISH painting consists of using DNA, highly enriched for sequences unique to a particular chromosome, labeled with a reporter molecule, such as a fluorochrome, and hybridizing it to metaphase chromosome spreads. At the same time, hybridization of any labeled repetitive sequences (common to all chromosomes) that may be present are blocked by competitive hybridization to unlabeled repetitive DNA. In this manner, stable aberrations such as translocations can be observed. FISH and the combinatorial derivatives of FISH, such as Spectral Karyotyping, are generally limited by their ability to detect only breaks, interchanges and numerical aberrations. Giemsa-banding, also known as G-banding, or similar approaches such as R- or Q-banding, is suitable only for detecting changes in banding patterns caused by chromosome inversions when the inversion involves a segment of the chromosome large enough to produce a recognizable change in the pattern of banding. While it may be possible with difficulty to detect an inversion with breakpoints near the midpoints of adjacent dark and light bands, many larger disruptions involving regions containing more than two or three bands might not always produce a recognizable change in these light/dark patterns of banding. Band lengths of fully condensed human mitotic chromosomes average ˜107 base pairs.
A chromatid is a replicated chromosome consisting of two identical parts that will be divided equally between daughter cells at mitoses when two new cells are created from one as cell populations grow. At mitosis, then, each chromosome consists of two identical chromatids and each of these consists of a linear, double-stranded DNA molecule. A strand of DNA is basically a phosphate deoxyribose polymer, each with one of four purine or pyrimidine base residues (A, T, G, or C) attached. Beginning with the first sugar there is a phosphate group at the 5′ position and a hydroxyl group at the 3′ position. This hydroxyl group is in turn joined to the next sugar at the 5′ position and the alternating chain continues until the other end of the linear strand where there is a 3′ hydroxyl group. The strands are associated by hydrogen bonding and are thus not covalently joined. The hydrogen bonding between the two strands occurs only between certain bases; that is, A with T and C with G. This results in what is known as complementary base pairing between the two opposite strands.
The genome of a cell must be replicated prior to the process of cell division in order to provide the same genetic information contained in the parent cell to each of the two new daughter cells. Before this replication, each chromosome consists of one double stranded DNA molecule, with one strand complementary to the other. During replication the complementary single strands of the chromosome are effectively separated, with each one becoming the basic part of a new chromatid. If one of these parental strands is oriented in the 5′→3′ direction along its length with respect to some arbitrary reference direction, then the 5′→3′ direction of the complementary strand will be oriented in the opposite direction. After replication the new synthesized strands each will likewise be complementary to its respective parental strand. The 5′→3′ direction of single strands within a double stranded DNA molecule is sometimes referred to as the polarity of the strand.
An inversion is an abnormality in chromosome structure that can result from, effectively, two double-stranded breaks occurring at different points along a portion of the chromosome, and rather than the breaks becoming rejoined in their original condition by cellular DNA repair processes, they occasionally rejoin incorrectly in such a way that this interstitial portion of the chromosome becomes effectively rotated through 180° after a “misrejoining” among the broken ends. Importantly, this misrejoining must occur in such a way as to maintain the same 5′→3′ polarity of the strands of the chromosome and that of the inverted segment. While the backbone polarity is maintained, the DNA sequence of the nitrogenous bases within the segment is reversed.
Chromosome ‘paints’ are mixtures of fluorescent DNA probes, or other types of molecular markers, highly enriched in sequences unique to a particular chromosome that allow a specific chromosome to be identified based on accepted cytogenetic practices that render the chromosome visible using a fluorescent microscope. Such probes can be purchased from a number of vendors.
The first complete draft of the human genome was made in 2000, and refinements have been made to the database since then. The GenBank database is made available to the public by the National Center for Biotechnology Information (NCBI) of the National Library of Medicine of the National Institutes of Health. Most of the DNA sequences have been ordered into contiguous sequences called contigs.
The CO-FISH technique, developed in the 1990s, permits fluorescent probes to be specifically targeted to sites on either chromatid, but not both. To date, this technique has been used almost entirely for detection of highly repetitive DNA which consists of a series of DNA sequences repeated over and over again, up to thousands of times and which contains few, if any, genes. Such regions are commonly found at sites on a chromosome involved in the mechanics of genome partitioning such as centromeres and telomeres. In “Strand-Specific Fluorescence in situ Hybridization: The CO-FISH Family” by S. M. Bailey et al., Cytogenet. Genome Res. 107: 11-14 (2004), chromosome organization is studied using strand-specific FISH (fluorescent or fluorescence in situ hybridization) [CO-FISH; Chromosome Orientation-FISH] which involves removal of newly replicated strands from the DNA of metaphase (mitotic) chromosomes, resulting in single-stranded target DNA. Each newly replicated double helix contains one parental DNA strand plus a newly synthesized strand, and it is this newly synthesized strand that is removed. When labeled single-stranded probes are hybridized to such targets, the resulting strand-specific hybridization is capable of providing previously unattainable cytogenetic information. Hybridization is a process in which two complementary nucleic acid sequences anneal by base pairing. In the context of FISH, “in situ” refers to hybridization of a nucleic acid sequence probe to the DNA of chromosomes, where the chromosomes are in cells that are attached to a glass microscope slide.
For example, it is known that mammalian telomeric DNA consists of tandem repeats of the (TTAGGG) sequence, oriented 5′ to 3′ towards the termini of all vertebrate chromosomes. Thus, CO-FISH with a suitable telomere probe reveals the absolute 5′ to 3′ orientation of DNA sequences relative to the chromosome's pter →qter direction (end of p or short arm of the chromosome to the end of the q or long arm of the chromosome).
The removal of the newly replicated strands using the CO-FISH procedure leaves the original (parental) strands largely intact. Thus, for the purposes of subsequent hybridization reactions, the two sister chromatids of a chromosome are rendered single stranded, and complementary to one another. The ability of CO-FISH to restrict hybridization of single-stranded probes to only one of the two sister chromatids means that it can also be used for inversion detection. Because an inversion reverses the orientation of the DNA sequences within the inversion region, it becomes visible as a jump or switch in probe signal from one chromatid to its sister chromatid. Such a switch can readily be detected when compared to a reference probe outside of the inverted region.